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Biomedical subjects

V N Kalinin

Publications and source records attributed to V N Kalinin.

At least 19 recordsLinked to original sources

Altered ratio of collagen chains in bone of a patient with non-lethal osteogenesis imperfecta.

Bone from a patient with osteogenesis imperfecta contained type III collagen which was absent in control bone. The ratio of alpha 1(I)/alpha 2(I) in type I collagen of patient's bone was increased (2.9 vs. 2.3 +/- 0.2 in controls) and the ratio of dimers beta 11/beta 12/beta 22 was altered due to the increased beta 22 content. No abnormality was observed in collagen from the patient's skin. The altered composition of collagen in bone, but the normal composition in skin suggests that the disease in the patient is due to impaired regulation of the synthesis of collagens in bone, rather than by a mutation in one of the two type I collagen genes. Unlike in skin, all the type III collagen in patient's bone was pepsin-soluble indicating an inability of the bone to incorporate type III collagen into mature highly cross-linked extracellular matrix.

Bone and Bones

Frequency of the F508 deletion in cystic fibrosis patients from the European part of the USSR.

The frequency of the F508 deletion (delta F508) has been analyzed in 189 cystic fibrosis (CF) patients from the European part of the USSR, viz. 127 northern Slavonians (Leningrad region), 30 southern Slavonians (the Ukraine), 10 central Slavonians (Moscow region), 14 Moldavians (Kishenev region) and 8 Lithuanians (Vilnius region). The distribution of CF+ chromosomes with and without delta F508 varied significantly in the different ethnic groups studied and correlated with the clinical manifestation of CF. The overall frequency of delta F508 in Slavonian patients is equal to 62.5%, approximately 90% of them being heterozygous or homozygous for this mutation. The frequency of the deletion among 99 Slavonian patients with severe disease manifestation (pancreatic insufficiency, PI) is equal to 67.5%, only 12 patients having pancreatic sufficiency (PS, 17.5%). The highest value of delta F508 (77.4%) is registered in PI/CF patients of the southern Slavonian group; it is much less frequent (about 57%) in relevant groups of Slavonians from the northern and central parts of the country. Unusually low frequencies (24% and 26%) of delta F508 are detected in a few samples of Lithuanian and Moldavian CF patients, respectively. All delta F508+CF-chromosomes of Slavonian origin are associated with haplotypes 2.2.2. defined by the restriction fragment length polymorphism sites KM19/PstI, CS.7/Hin6I and MP6d-9/MspI, although a high proportion (about 25%) of unknown mutations is associated with the same haplotype. Haplotype B (allele 1XV2c/TaqI; allele 2 KM19/PstI) accounts for 91% of delta F508+CF chromosomes. Our data are consistent with the hypothesis of a single origin and subsequent diffusion of this major CF mutation; however, its interpopulational dissemination in Eastern Europe does not follow the suggested south-east to north-west gradient in Western Europe. The significance of these data for prenatal diagnosis and carrier screening of CF mutations is briefly discussed.

Chromosome Deletion

Restriction fragment length polymorphisms of three collagen genes in Russians and Buryats.

The restriction fragment length polymorphisms (RFLPs) of 3 collagen genes have been studied in two USSR populations. Four high-frequency alleles, also reported in other populations, were observed. The frequencies of alleles for the polymorphic sites MspI and EcoRI of the COL1A2 gene, HindIII of the COL2A1 gene and EcoRI of the COL3A1 gene were found to be 0.836, 0.398, 0.447 and 0.786, respectively, in Russians from Moscow (Caucasians), and 0.883, 0.519, 0.648 and 0.809, respectively, in Buryats from the Baikal region (Mongoloids). The RFLPs showed a fair agreement with the Hardy-Weinberg equilibrium. The frequencies were compared with those of other populations.

Alleles

Preliminary results on the frequency of the delta F508 mutation in cystic fibrosis patients from the USSR.

The frequency of the delta F508 mutation in a sample of 29 cystic fibrosis (CF) families from the USSR is equal to 44.8%, in agreement with previously reported haplotype data. If confirmed on a larger sample of CF patients from the USSR, this result might be indicative of the presence of a larger heterogeneity of CF mutations in the Soviet population than in other European populations.

Cystic Fibrosis

Modified method for peptide mapping of collagen chains using cyanogen bromide-cleavage of protein within polyacrylamide gels.

A highly efficient method for cyanogen bromide (CNBr)-mapping of collagen peptides is described. This method was developed based on polypeptide cleavage by CNBr within gel slices according to Barsh et al. [1981) Collagen Relat. Res. 1, 543-548). The proposed method has the following advantages: (i) Analysis of both radiolabeled and unlabeled collagens is possible; (ii) CNBr-cleavage of polypeptides is performed in gel pieces which contain individual bands; (iii) The peptide losses are minimized, offering a more complete analysis of collagens including the low molecular weight CNBr-peptides.

Collagen

The absence of type II collagen and changes in proteoglycan structure of hyaline cartilage in a case of Langer-Saldino achondrogenesis.

Structural analysis of hyaline cartilage extracellular matrix components from the ribs and knee joint of a stillborn female with type II achondrogenesis was carried out. The absence of type II collagen, a decrease in the amount of proteoglycans (PG), and structural changes in PG, namely, increased electrophoretic mobility of PG, lower relative content of chondroitin 4-sulfate (Ch4-S), lower molecular weight and decreased total chondroitin sulfate (ChS) sulfation, were detected. Increased amounts of type I and type III collagens, atypical for hyaline cartilage, were revealed. Among the link proteins (LPs), a large protein with a mol. wt. of 48 kDa was predominant. Molecular and cellular mechanisms of the pathogenesis of achondrogenesis ("chondrogenesis imperfecta") are discussed. The data obtained suggest that the primary defect in type II achondrogenesis involves ChS or type II collagen synthesis.

Achondroplasia

[Isolation of high molecular weight eukaryotic DNA with the use of potassium acetate].

The simple technique for isolation of high-molecular eucaryotic DNA has been proposed. It includes cell or nuclei lysates by sodium dodecylsulfate in the presence of pronase, proteins precipitation by potassium acetate, DNA precipitation by ethanol. The DNA isolated by this technique is easily cleaved by restriction endonucleases and can be used for analysis of the unique genes by blot-hybridization. The yield of DNA is similar or somewhat higher than that in case of using the standard methods including phenol extraction or phenol-chloroform extraction.

Acetates

[Optimization of the method of cyanogen bromide mapping of polypeptides separated by polyacrylamide gel electrophoresis].

Two highly efficient methods of CNBr-peptide mapping of polypeptides divided by polyacrylamide gel electrophoresis are described. The first is elaborated on the basis of peptide mapping of collagen proposed by G. Barsh et al. The following three modifications diminish wasting the material essential for the method. 1. CNBr treatment takes place in the absence of CNBr solution outside the gel, excluding the peptides elution from the gel fragments in the process of mapping. 2. After CNBr treatment the solution of CNBr is substituted by the samples buffer before electrophoresis by means of drying and subsequent addition of minimal volumes of the buffer. The latter procedures substitute the gel washing out by the buffer solution. 3. The step of washing the gel fragments by the 70% strong solution of formic acid before CNBr treatment is excluded. The second method of CNBr-peptide mapping is notable for extracting peptides from the gel fragments in the process of CNBr-treatment and permits obtaining of the high quality peptide electrophoregrams.

Collagen

[Isolation of a restriction endonuclease with HindIII-specificity from Bordetella bronchiseptica].

Site-specific restriction endonuclease BbrI has been found in bacteriophage resistant strain B. bronchioseptica 4994. The technique was elaborated for purification of BbrI to the stage free of nuclease and phosphatase contamination. The yield of purified enzyme is 6000-20 000 units per 10 g of biomass. BbrI recognises and cleaves the same DNA sequence as HindIII with the formation of four-nucleotide cohesive ends. The simplicity of cultivation, security for human, presence of the single restriction endonuclease and the high level of its production make B. bronchioseptica 4994 a promising producer of BbrI restriction endonuclease, isoshizomeric to HindIII, for use in experimental practice in industry.

Bordetella

[Limitation of phage multiplication by microbes of the genus Bordetella].

Possible causes limiting the multiplication of Bordetella phages or inducing their restriction, such as the influence of lysogenic immunity and the restriction-modification (R-M) system or the incompatibility of the receptor apparatus, have been studied. The limitation of the multiplication of phages by some B. bronchiseptica and B. pertussis strains has been shown to be due to the presence of the R-M system and lysogenic immunity. In five B. bronchiseptica strains and two B. pertussis strains site-specific endonucleases (restrictases) with Hind III specificity have been detected. One B. bronchiseptica strain without the R-M system has been detected. B. bronchiseptica strains producing site-specific endonucleases are practically nonpathogenic for humans, grow in common culture media and selectively produce only one restrictase, type Hind III, which guarantees from the admixture of other specific endonucleases. The B. parapertussis strains under study (altogether 100 strains) have not been found to limit the multiplication of Bordetella test phages. The absence of site-specific endonucleases has also been confirmed biochemically. These strains are recommended as indicator strains for the multiplication of Bordetella phages.

Adsorption

Construction and properties of hybrid plasmids carrying the E. coli gal operon.

A family of hybrid plasmids carrying the entire gal operon of E. coli and designated pgal was constructed in vitro. In the case of pgal 1 (mol. wt. 16.4 Md), a fragment cut by Bam HI endonuclease from lambda gal phage DNA (lambda D-J-gal-att-int) was joined to pMB9 and cloned in the gal-strain of E. coli, which was grown on selective media with galactose as a sole source of carbon. Plasmid pgal2 was derived from pgal 1 by elimination of the 1.1 Md fragment located between the two EcoRI sites and carrying the lambda att-int region and part of pMB9. To obtain pgal3, the 10.7 Md fragment of lambda DNA located between the two SmaI sites (lambda D-J and part of pMB9) in pgal2 was cut out and the resulting flush-end fragments were sealed by the T4DNA ligase. The mol. wt. of pgal3 containing one SmaI site amounted to 4.6 Md, while several pgal3 variants that had lost their SmaI site were still smaller. Plasmid pgal1 inhibited the growth of the gal- host cells, which effect could be overcome by the accompanying helper pMB9. The presence of pgal2 and pgal3 supported the growth and multiplication of gal- cells on selective media even without the helper plasmid. The total amount of pgal plasmid DNA per cell was constant and equalled 60--70 Md (4 copies of pgal1 or 15--16 copies of pgal3, ColE1 or pMB9). This might explain why the co-presence of pMB9 helper does alleviate the "harmful" effects of the plasmid pgal1 (which carries att-int genes), by reducing the copy number of the latter from four to one.

Bacteriophage lambda